Molecular characterisation and expression analysis of SEREX-defined antigen NUCB2 in gastric epithelium, gastritis and gastric cancer

NUCB2 is an EF-hand Ca2+ binding protein that has been implicated in various physiological processes like calcium homeostasis, hypothalamic regulation of feeding and TNF receptor shedding. In our previous study we identified NUCB2 as a potential tumour antigen eliciting autoantibody responses in 5.4% of gastric cancer patients but not in the healthy individuals. The current study aimed to elucidate the molecular mechanism underlying NUCB2 immunogenicity and to gain an insight into the physiological functions of NUCB2 in the stomach. mRNA expression analysis demonstrated that NUCB2 is ubiquitously expressed in normal tissues, including lymphoid tissues, and downregulated in gastric tumours when compared with the adjacent relatively normal stomach tissues. The search for molecular alterations resulted in the identification of novel mRNA variants transcribed from an alternative promoter and expressed predominantly in gastric cancers. Western blot analysis demonstrated that the protein levels correspond to mRNA levels and revealed that NUCB2 is phosphorylated in gastric mucosa. Furthermore, a 55 kDa isoform, generated presumably by yet an unidentified post-translational modification was detected in gastric tumours and AGS gastric cancer cells but was absent in the relatively normal gastric mucosa and thereby might have served as a trigger for the immune response against NUCB2. Staining of stomach tissue microarray with anti-NUCB2 antibody revealed that it is expressed in the secretory granules of chief cells and in the cytoplasm of parietal cells in the functioning gastric glands which are lost in atrophic glands and tumour cells. Hence we propose that NUCB2 may be implicated in gastric secretion by establishing an agonist-releasable Ca2+ store in ER or Golgi apparatus, signalling via heterotrimeric Gα proteins and/or mediating the exocytosis of the secretory granules

Serological identification and expression analysis of gastric cancer-associated genes

Serological identification of tumour antigens by recombinant expression cloning has proved to be an effective strategy for the identification of cancer-associated genes having a relevance to cancer aetiology and progression, and for defining possible targets for immunotherapeutic intervention. In the present study we applied this technique to identify immunogenic proteins for gastric cancer that resulted in isolation of 14 distinct serum-reactive antigens. In order to evaluate their role in tumourigenesis and assess the immunogenicity of the identified antigens, we characterised each cDNA clone by DNA sequence analysis, mRNA tissue distribution, comparison of mRNA levels in cancerous and adjacent non-cancerous tissues and the frequency of antibody responses in allogeneic patient and control sera. Previously unknown splice variants of TACC1 and an uncharacterised gene Ga50 were identified. The expression of a newly identified TACC1 isoform is restricted to brain and gastric cancer tissues. Comparison of mRNA levels by semi-quantitative RT–PCR revealed a relative overexpression of three genes in cancer tissues, including growth factor granulin and Tbdn-1 – an orthologue of the mouse acetyltransferase gene which is associated with blood vessel development. An unusual DNA polymorphism – a three-nucleotide deletion was found in NUCB2 cDNA but its mRNA level was consistently decreased in gastric tumours compared with that in the adjacent non-cancerous tissues. This study has revealed several new gastric cancer candidate genes; additional studies are required to gain a deeper insight into their role in the tumorigenesis and their potential as therapeutic targets

Voltage-dependent anion-selective channel (VDAC) interacts with the dynein light chain Tctex 1 and the heat-shock protein PBP74

The voltage-dependent anion-selective channel 1 (VDAC1), i.e. eukaryotic porin, functions as a channel in membranous structures as described for the outer mitochondrial membrane. the cell membrane, endosomes, caveolae, the sarcoplasmatic reticulum, synaptosomes. and post-synaptic density fraction. The identification of VDAC1 interacting proteins may be a promising approach for better understanding the biological context and function of the channel protein. In this study human VDAC1 was used as a bait protein in a two-hybrid screening, which is based on the Sos recruitment system (SRS). hVDAC1 interacts with the dynein light chain Tctex-1 and the heat-shock protein peptide-binding protein 74 (PBP74)/mitochondrial heat-shock protein 70 (mtHSP70)/glucose- regulated protein 75 (GRP75)/mortalin in vivo. Both interactions were confirmed by overlay-assays using recombinant partner proteins and purified hVDAC1. Indirect immunofluorescence on HeLa cells indicates a co-localisation of hVDAC1 with the dynein light chain and the PBP74. In addition, HeLa cells were transfected transiently with enhanced green fluorescent protein (EGFP)-hVDAC1 fusion proteins, which also clearly co-localise with both proteins. The functional relevance of the identified protein interactions was analysed in planar lipid bilayer (PLB) experiments. In these experiments both recombinant binding partners altered the electrophysiological properties of hVDAC1, While rTctex-1 increases the voltage-dependence of hVDAC1 slightly, the rPBP74 drastically minimises the voltage-dependence, indicating a modulation of channel properties in each case. Since the identified proteins are known to be involved in the transport or processing of proteins, the results of this study represent additional evidence of membrane-associated trafficking of the voltage-dependent anion-selective channel 1. (C) 2002 Elsevier Science Ltd. All rights reserved